System and method for processing bone marrow

ABSTRACT

A system for collecting and processing donated bone marrow comprises a closed and/or sealable collection container and a closed and/or sealable satellite container. The system may also include at least one porous medium interposed between the collection container and the satellite container. The porous medium is a size gradient depletion media. The system may also include at least one third container in fluid communication with the collection container. The system may also include at least one manifold interposed in the fluid flow path between the collection container and the third container.

TECHNICAL FIELD

[0001] This invention relates to a system for processing bone marrowdonated for the purpose of therapeutic transfusion, for separating thebone marrow composition into one or more constituents, and,particularly, to improved methods and apparatuses for preparing andprocessing a bone marrow composition. This invention also relates to abiological fluid processing system for processing biological fluid intoits various components.

BACKGROUND OF THE INVENTION

[0002] Bone marrow is a spongy tissue found inside bones. The bonemarrow in the breast bone, skull, hips, ribs, and spine contains stemcells. Stem cells produce the body's blood cells, e.g., leukocytes, thatfight infection); erythrocytes, that carry oxygen to and remove wasteproducts from organs and tissues; and platelets, that enable the bloodto clot.

[0003] Bone marrow, and some of the cells and cell types that arepresent in bone marrow, have been found to have significant therapeuticefficacy in the treatment of a number of diseases and conditions. Forexample, stem cells may be separated from the bone marrow compositionand may be used in the treatment of many diseases. Filtered marrowitself may be used as a transplant graft in the treatment of leukemia,aplastic anemia, various lymphomas (e.g., Hodgkin's disease), multiplemyeloma, certain immunodeficiencies, and a variety of cancers (typicallysolid tumors, such as breast and ovarian cancer).

[0004] The development of plastic blood collection bags has facilitatedthe separation of donated bone marrow into its various components andanalogous products, thereby making these different components availableas a transfusion product.

[0005] Further, significant improvements in reducing the immunogenicityof bone marrow transplanted into a patient has greatly increased the useof bone marrow as a competent and desirable therapeutic composition.

[0006] For these and other reasons, harvesting bone marrow andseparating the bone marrow into components has substantial therapeuticand monetary value. This is nowhere more evident than in treating theincreased damage to a patient's immune system caused by the higher dosesand stronger drugs now used during chemotherapy for cancer patients.These more aggressive chemotherapy protocols are directly implicated inthe reduction of the platelet content of the blood to abnormally lowlevels.

[0007] A conventional bone marrow collection procedure may include thefollowing:

[0008] (1) Under general anesthesia, a bone marrow aspiration needle isinserted into the iliac crest (the cavity of the rear hip bone) where alarge quantity of bone marrow is located. The bone marrow is extractedwith a needle and syringe. Several skin punctures on each hip andmultiple bone punctures are usually required to extract the requisiteamount of bone marrow.

[0009] (2) About 10-30 cc of bone marrow are drawn from each bonepuncture, and although the total amount drawn is variable (dependingprimarily on the size of the donor and the concentration of the bonemarrow cells), usually about 1 liter and up to 1.5 liters of bone marroware harvested.

[0010] (3) Each syringe of bone marrow drawn from the donor is thenindividually expressed into an open collection bag or an open container.The collection bag typically includes an anti-coagulant solution such asheparin and/or CPDA-1.

[0011] (4) The used syringe is then used to draw anti-coagulant solutioninto the syringe, the solution is expelled, and the syringe is usedagain to draw bone marrow. The process is repeated 30-50 times or moreuntil up to about 1.5 liters of bone marrow is harvested.

[0012] (5) During or near the end of the collection process, a sample ofabout 5 cc is then drawn from the collection bag or container, and theamount of stem cells in the sample is either determined or inferred.Harvesting is usually completed when the sample contains about 1-3×10⁸nucleated cells per kilogram of body weight of the recipient.

[0013] (6) The harvested bone marrow composition is then filtered,generally within about 6 to 8 hours of harvesting. The filtrationprocess usually involves a series of filters having different pore sizeratings, typically 850μ, 500μ, and 200μ. These filters remove bonefragments, microaggregates, blood clots, and other undesirable debrisfrom the bone marrow composition.

[0014] (7) The filtered bone marrow is then processed according to itsend use. For example, if the bone marrow will be used in an autologoustransplant, the filtered bone marrow will be frozen (cryopreserved) andstored at a temperature between about 80° C. and about −196° C. untilthe day of the transplant. For allogeneic transplants, the bone marrowwill be treated to remove T-cells, then transferred directly to aninfuser for administration to the patient.

[0015] The existing regimen for harvesting bone marrow is time-consumingand costly. Further, the present state of the art involves the use ofopen containers and/or open systems.

[0016] In view of this, there is a growing need for an efficient systemand method for collecting and processing bone marrow, and for harvestingbone marrow in a closed or sealed system.

[0017] The devices and methods of this invention alleviate theabove-described problems and, in addition, provide a higher yield ofsuperior quality bone marrow.

[0018] A problem attendant with the separation of various blood and bonemarrow components using a multiple bag system is that the highlyvaluable components become trapped in the conduits connecting thevarious bags and in the various devices that may be used in the system.Conventional processing and storage techniques contribute to theseproblems. For example, air, in particular oxygen, present in storedblood and blood components, or in the storage container, may lead to animpairment of the quality of the blood components, and may decreasetheir storage life. More particularly, oxygen may be associated with anincreased metabolic rate (during glycolysis), which may lead todecreased storage life, and decreased viability and function of wholeblood cells. For example, during storage red blood cells metabolizeglucose, producing lactic and pyruvic acids. These acids decrease the pHof the medium, which in turn decreases metabolic functions. Furthermore,the presence of air or gas in the satellite bag may present a risk whena patient is transfused. For example, as little as 5 ml of air or gasmay cause severe injury or death. Despite the deleterious effect ofoxygen on storage life and the quality of bone marrow blood and bonemarrow components, the prior art has not addressed the need to removegases from bone marrow processing systems during collection andprocessing.

[0019] Because of the high cost and limited availability of bone marrowcomponents, a device comprising a porous medium used to depleteleucocytes from biological fluid should deliver the highest possibleproportion of the component present in the donated bone marrow. An idealdevice for the filtering or leucocyte depletion of bone marrow would beinexpensive, relatively small, and be capable of rapidly processing thecomponents obtained from about one unit or more of biological fluid(e.g., donated bone marrow), in, for example, less than about one hour.Ideally, this device would reduce the leucocyte content to the lowestpossible level, while maximizing the yield of a valuable blood componentwhile minimizing an expensive, sophisticated, labor intensive effort bythe operator of the system. The yield of the bone marrow or a componentshould be maximized while at the same time delivering a viable andphysiologically active component. It may also be preferable that thebone marrow filter or porous medium be capable of removing platelets, aswell as fibrinogen, fibrin strands, tiny fat globules, and othercomponents such as microaggregates which may be present in the bonemarrow.

[0020] Definitions

[0021] The following definitions are used in reference to the invention:

[0022] (A) Bone marrow or Biological Fluid is the soft tissue withinbone cavities, and typically contains whole blood, hematopoieticprecursor cells and hematopoietic cells that are maturing intoerythrocytes, five types of leukocytes, and thrombocytes. Harvested bonemarrow typically includes these components, as well as bone chips,megakaryocytes, stem cells, fat globules, blood clots, fibrin,platelets, among other biological and/or cellular matter. Bone marrow orbiological fluid also includes any treated or untreated fluid associatedwith living organisms, particularly bone marrow, including harvestedunseparated (whole) bone marrow, warm or cold bone marrow, cryopreservedbone marrow, and stored or fresh bone marrow, treated bone marrow, suchas bone marrow diluted with a physiological solution, including but notlimited to saline, nutrient, and/or anticoagulant solutions; one or morebone marrow components, such as stem cells; and analogous bone marrowproducts derived from bone marrow or a bone marrow component. Thebiological fluid may include leucocytes, or may be treated to removeleucocytes. As used herein, bone marrow product or biological fluidrefers to the components described above, and to similar bone marrowproducts or biological fluids obtained by other means and with similarproperties. In accordance with the invention, each of these bone marrowproducts or biological fluids is processed in the manner describedherein.

[0023] A typical harvesting procedure commonly draws up to about 1.5liters of a composition containing bone marrow from the donor into a bagwhich contains an anticoagulant to prevent the bone marrow fromclotting. However, the amount drawn differs from patient to patient anddonation to donation.

[0024] (B) Filtered bone marrow: refers to a composition containing bonemarrow and stem cells that is suitable for use as a transplant graft oran organ graft. The bone marrow harvested from a donor is treated andprocessed using various regimens before it is suitable for use as atransplant graft. For example, the harvested bone marrow may be mixedwith a nutrient and/or anti-coagulant solution, and may be filtered toremove debris and the like.

[0025] (C) Porous medium: refers to the porous medium through which oneor more bone marrow, bone marrow components, or biological fluids pass.A typical porous medium is a filter for removing undesirableconstituents, such as pieces of bone, microaggregates, blood clots, andthe like, from the bone marrow. The bone marrow processing system mayoptionally include a leucocyte depletion filter or porous medium, whichrefers generically to any one of the media which deplete leucocytes fromthe bone marrow or a bone marrow component.

[0026] The porous media according to the invention may be connected to aconduit interposed between the containers, and may be positioned in ahousing which in turn can be connected to the conduit. As used herein,filter assembly refers to the porous medium positioned in a suitablehousing. An exemplary filter assembly may include pore size filterassembly and/or a leucocyte depletion assembly or device. A biologicalfluid processing system, such as a bone marrow collection and processingsystem, may comprise porous media, preferably as filter assemblies.

SUMMARY OF THE INVENTION

[0027] In the devices and methods of the present invention, bone marrowis harvested from a donor and collected or pooled in a collection bag.Typically, the bone marrow is filtered at the time of processing, which,in the United States, is generally within about 6 to 8 hours of the timethe bone marrow is harvested. Thus, as a biological fluid is transferredfrom the collection bag, debris and other undesirable constituents maybe removed by the appropriate porous medium, and filtered bone marrow iscollected in the satellite bag.

[0028] In accordance with the invention, a system is provided whereby abiological fluid such as bone marrow or a bone marrow composition isprocessed to form a bone marrow composition suitable for transplantationinto a recipient, or is processed into one or more of its components. Anapparatus and system according to the invention includes a closedcollection container having at least one access port and a firstconnector for communicating with a second, transfer, or satellitecontainer; and a transfer or satellite bag configured to establish fluidflow between the first container and the second container.

[0029] Some embodiments of the invention also include at least onefilter assembly interposed in the fluid flow path between the firstcontainer and the second container. The filter assembly may beconfigured to establish and receive fluid flow from the collectioncontainer, said filter assembly having at least one porous mediumthrough which bone marrow passes, said filter assembly may also beconfigured to establish fluid flow into a transfer or satellite bag.

[0030] Some embodiments of the invention also include at least one thirdcontainer, preferably in fluid communication with the first container.Typically, the third container will include an anti-coagulant solution,a nutrient solution, a rinsing solution, a priming solution, or thelike.

[0031] Some embodiments of the invention also include at least onemanifold interposed between the first container and the secondcontainer. The manifold may be configured to establish a fluid flow pathinto the first container, into the third container, and/or between thefirst container and the second container.

[0032] for transferring the harvested bone marrow from the syringe tothe collection bag. In preferred embodiments of the invention, themanifold includes one or more ports for transferring bone marrow,harvested from the donor in a harvesting container (typically asyringe), into the first or collection container.

[0033] Additionally, processes and systems according to the inventionmay include a gas outlet that allows gas that may be present in thesystem out of the system or a component of the system.

[0034] Processes and systems according to the invention may also includea gas inlet that allows gas into the system to recover a biologicalfluid that may be entrapped or retained during processing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is an embodiment of a biological fluid processing systemaccording to the invention.

[0036]FIG. 2 is an embodiment of a manifold for transferring bone marrowinto a processing system according to the invention.

SPECIFIC DESCRIPTION OF THE INVENTION

[0037] The present invention involves a bone marrow collection andprocessing assembly or system comprising a closed or sealed firstcontainer and a closed or sealed second container, and a conduitinterconnecting the first container with the second container, saidfirst container having at least one access port.

[0038] Some embodiments of the invention also include at least one firstporous medium interposed in the fluid flow path between the firstcontainer and the second container. The porous medium, preferably housedin a chamber to form a filter assembly, may include a microaggregatefilter element and/or a gel pre-filter element. In a preferredembodiment of the invention, the system includes at least onemicroaggregate filter having a pre-determined pore size; in a mostpreferred embodiment of the invention, the system includes at least onemicroaggregate filter having a pore size selected from 850μ, 500μ, and200μ, or combinations thereof.

[0039] Some embodiments of the invention also include at least one thirdcontainer in fluid communication with the first container. Typically,the third container will include an anti-coagulant solution, a nutrientsolution, a rinsing solution, a priming solution, or the like. It isintended that the invention should not be limited by the type or use ofsolution in the third container.

[0040] Some embodiments of the invention also include at least onemanifold interposed in the fluid flow path between the first containerand the third container. The manifold may be variously configured andmulti-functional. For example, the manifold may include at least oneport adapted to engage a portion of the harvesting container, said portbeing configured to establish a fluid flow path between the harvestingcontainer and the first container. The port may also be configured toestablish a fluid flow path between the harvesting container and thethird container. For example, it may be desirable to transfer a bonemarrow composition in a syringe (e.g., a harvesting container) to thefirst container or a collection container, and for drawing andexpressing anti-coagulant solution into and out of the syringe.

[0041] In the embodiments of the invention that include a manifold, themanifold may be variously configured. In accordance with the presentinvention, the manifold may be configured or adapted to provide one ormore of the following: a fluid flow path between the harvestingcontainer and the collection container, a fluid flow path between theharvesting container and a third container, a fluid flow path betweenthe third container and the collection container, or combinationthereof. In a preferred embodiment of the invention, the manifold isconfigured to provide a individual fluid flow paths between theharvesting container, the collection container, and the third container.

[0042] A system according to the invention also includes one or moreflow restrictors so that the collection container, the filter assembly,the transfer container, the third container, the manifold, orcombinations thereof may be separated from the system.

[0043] The bone marrow collection and processing system may also includeone or more fluid flow valves for opening and/or closing a fluid flowpath anywhere in the system, or for channeling fluid through a specificfluid flow path. Exemplary fluid flow valves include but are not limitedto a stopcock, a plunge valve, and a re-sealable septum valve. The fluidflow valves may by unidirectional or multi-directional. In some of thefluid flow valves, it may be desirable that the valve is re-sealable orclosable.

[0044] As shown in more detail below, the assembly may also includeadditional containers, porous media, and conduits interconnecting thevarious elements of the system or assembly.

[0045] The invention also involves a method for collecting andprocessing bone marrow comprising harvesting bone marrow from a donor,collecting the bone marrow in a first closed or sealed container;passing the bone marrow through at least one porous medium, the porousmedium comprising a microaggregate filter having a predetermined poresize or rating; and passing the filtered bone marrow into a secondclosed or sealed container. In a preferred embodiment of the invention,the porous medium is positioned in a housing to form a filter assembly,and the filter assembly is interposed in the fluid flow path between thefirst container and the second container. In a most preferred embodimentof the invention, multiple filter assemblies are interposed in the fluidflow path between the first container and the second container, eachfilter assembly having a porous medium of different pore rating than theother porous media. In general, harvested bone marrow is processed orfiltered as soon as practicable after donation in order to moreeffectively reduce or eliminate contaminating factors, including but notlimited to leucocytes and microaggregates.

[0046] The invention also involves a method for collecting andprocessing bone marrow comprising harvesting bone marrow from a donorinto a harvesting container, such as a syringe or the like; transferringthe harvested bone marrow into a closed or sealable collectioncontainer; repeating the harvesting step and the transferring step untila sufficient quantity of bone marrow and/or stem cells are obtained, andcollecting the bone marrow in the collection container. The method mayfurther include passing the harvested bone marrow in the first containerthrough a first porous medium, the first porous medium comprising amicroaggregate filter having a pre-determined pore size or rating; andpassing the filtered bone marrow into a second closed or sealablecontainer.

[0047] In a preferred embodiment of the invention, transferring theharvested bone marrow into a collection container may include passingthe bone marrow through a first fluid flow path into the collectioncontainer, closing the first flow path, opening a second fluid flow pathbetween a third container containing anti-coagulant solution and theharvesting container; passing anti-coagulant solution into theharvesting container; expressing anti-coagulant solution from theharvesting container, preferably into the third container; closing thesecond flow path; and repeating the harvesting step and the transferringstep until a sufficient quantity of bone marrow and/or stem cells areobtained.

[0048] In a most preferred embodiment of the invention, the transferringstep includes repeating the harvesting step and the transferring stepuntil a sufficient quantity of bone marrow and/or stem cells areobtained, then opening a fluid flow path between the first container andthe third container, and expressing the contents of the third containerinto the first container.

[0049] In a preferred embodiment of the invention, the bone marrowcollection and processing system is sealed and/or closed.

[0050] An exemplary bone marrow or biological fluid collection andprocessing system is shown in FIG. 1. The bone marrow processing systemis generally denoted as 10. It may comprise a first container orcollection bag 11, said first container comprising at least one accessport 12 and at least one discharge port 13; a second container (firstsatellite bag) 14; and, interposed between the first container 11 andthe second container 14, at least one filter assembly 15. Each of theassemblies or containers may be in fluid communication through tubing,preferably flexible tubing, 20, 21, and 22. In accordance with theinvention, there is a first fluid flow path between the first container11 and second container 14. A seal, valve, clamp 23, or transfer legclosure or cannula (not illustrated) may also be positioned in or on thetubing or in the collection and/or satellite bags. The seal (or seals)is opened when fluid is to be transferred between bags.

[0051] In a preferred embodiment of the invention, first container 11also includes at least one valve 24, preferably in fluid communicationwith access port 12. In the illustrated embodiment, valve 24 is in fluidcommunication with port 12 through conduit 22. Valve 24 permits accessto the collection bag; in a preferred embodiment of the invention, valve24 is or includes a connector adapted for communication with a syringeor the like, e.g., a Luer lock.

[0052] An apparatus or system according to the invention may alsoinclude one or more manifolds 30. Manifold 30 may be variouslyconfigured to provide closed and/or sealed fluid communication between aharvesting container (not shown) and first or collection container 11,to provide closed and/or sealed fluid communication between a harvestingcontainer (not shown) and third container 34, to provide closed and/orsealed fluid communication between first container 11 and thirdcontainer 34, to provide fluid communication between first container 11and a sampling container (not shown), such as a syringe, or combinationsthereof. An embodiment of the invention is illustrated in FIG. 2.

[0053] In FIG. 2, manifold 30 is in fluid communication with firstcontainer 11 through conduit 22. Manifold 30 includes port 31 configuredto engage complementary structures on a harvesting container (e.g., asyringe, not shown). In a preferred embodiment of the invention, theharvesting container includes a connector or the like suitable forengaging port 31. Such connectors are well known in the art, and includebut are not limited to a Luer lock.

[0054] Manifold 30 may include a channel, conduit 32, or the like, thatdefines a fluid flow path between the harvesting container and thecollection container. Manifold 30 may include a channel, conduit 33, orthe like, that defines a fluid flow path between the harvestingcontainer and a third container 34. Third container 34 preferablycontains an anti-coagulant solution, such as heparin, CPDA-1, DMSO, orthe like. Third container 34 may be directly attached to the manifold,may itself include structures that function as a manifold as definedherein, or may be connected to the manifold 30 through conduit 35.Manifold 30 may include a channel, conduits 32 and 33, or the like, thatdefines a fluid flow path between the first container 11 and the thirdcontainer 34.

[0055] In a system according to the invention, the flow path between thefirst container and the harvesting container is the second fluid flowpath; between the harvesting container and the third container, thethird fluid flow path; and between the first container and the thirdcontainer, the fourth fluid flow path.

[0056] In a preferred embodiment of the invention, the second, thirdand/or fourth fluid flow paths may include one or more valves,preferably in fluid communication with access port 31. The valves may bean element integral to a container or manifold, or may be connected to acontainer or manifold. In the illustrated embodiment, valve 36 isinterposed in the fluid flow path between first container 11 andmanifold 30, and valve 37 is interposed in the fluid flow path betweenthird container 34 and manifold 30. In accordance with the invention,valves 36 and 37 may be any valve, clamp, restrictor, diaphragm,connector, or the like that selectively permits fluid flow therethrough,and is suitable for use with a composition containing bone marrow and/oranti-coagulant solution. In a preferred embodiment of the invention,valves 36 and 37 are plunge valves, valves that open a channel when theplunger is depressed. Suitable valves are commercially available underthe tradename TRAC™ Valve.

[0057] As noted above, a system according to the invention alsopreferably includes one or more sampling ports. For example, ports 12,13, and 31 may function as sampling ports. In a preferred embodiment ofthe invention, first container 11 includes sampling port 40. In a mostpreferred embodiment of the invention, the sampling port maintains theassembly or system in a closed and/or sealed condition. For example,sampling port 40 may include a re-sealable septum or diaphragm or thelike that permits the insertion of a needle into the first container,and re-seals the first container when the needle is withdrawn.

[0058] One skilled in the art will recognize that multiple harvestingcontainers and/or multiple manifolds may be incorporated into a systemaccording to the invention.

[0059] In accordance with the present invention, any number andcombinations of assemblies, porous media, containers, conduits, andmethods of operation or processing are suitable. The invention comprisesstructures and methods for harvesting a composition comprising bonemarrow, transferring the harvested bone marrow to a collectioncontainer, and processing the collected bone marrow, more preferablyinto a composition containing bone marrow suitable for use as atransplant graft. One skilled in the art will recognize that theinvention as described here may be reconfigured into differentcombinations, elements, and processes which are included within thescope of the invention.

[0060] A method according to the present invention includes harvesting acomposition comprising bone marrow, passing the bone marrow through asystem according to the present invention, and processing the bonemarrow into a composition suitable for a pre-determined end use, e.g.,suitable as a transplant graft, as a source of stem cells, or as acomposition suitable for use as part of research and development relatedto one or more constituents in the bone marrow composition. As will bereadily apparent to one skilled in the art, methods, steps, or aspecific sequence of steps according to the invention will be dictatedin part by the specific elements incorporated in a specific system. Forexample, methods relating to a system lacking a third container wouldnot include steps that involve the third container.

[0061] The following provides exemplary methods and specific exemplarysteps to illustrate the function of an apparatus and system according tothe present invention.

[0062] Once a donor is prepared for a bone marrow harvest procedure,bone marrow from the donor may be drawn into a harvesting container,such as a syringe. Typically, about 5 cc to about 30 cc of bone marroware drawn. The harvesting container is then disconnected from the donorand attached to an apparatus according to the present invention. Forexample, a harvesting syringe may be connected to access port 12 onfirst or collection container 12 or on access port 31 on manifold 30.Collection container 12 typically includes a pre-determined amount ofanti-coagulant solution, and the harvested bone marrow is mixed with theanti-coagulant solution. In use, a biological fluid, e.g., bone marrow,is fed under sufficient pressure into the inlet of container 11 from anysuitable source of the biological fluid. For example, bone marrow may beharvested from a donor using a syringe or the like, and the bone marrowmay be injected from a syringe into the inlet of the collectioncontainer 11.

[0063] In the embodiments of the invention that include a manifold 30,the harvested bone marrow may be expressed or passed from the harvestingcontainer through port 31, through open valve 36, through conduit 22,and into collection bag 11. Valve 36 is then closed, valve 37 is thenopened, and anti-coagulant solution in third container 34 may beexpressed or drawn into the harvesting container through conduit 35,valve 37 conduit 33, and port 31. Once the harvesting container isflushed or rinsed with anticoagulant solution, the solution may bereturned to third container 34, and the harvesting container is readyfor re-use, i.e., for drawing another 5-30 cc of bone marrow from thepatient.

[0064] The harvesting step and the transfer of the bone marrowcomposition to the collection container may be repeated as often asdesired, typically until up to about 1.5 liters or more of bone marrowis harvested.

[0065] Once the desired amount of bone marrow is harvested, if thesystem includes a third container 34, the contents of third container 34(typically including a mixture of anti-coagulant solution and bonemarrow rinsed from the harvesting container), may be expressed or passedinto first container 11, where it is mixed with the bone marrowcomposition collected in first container 11. In the embodiment of theinvention shown in FIGS. 1 and 2, a fluid flow path is opened betweenthird container 34 and first container 11, typically by opening valve37, closing port 31, opening valve 36, and opening port 12. Thecomposition in first container 11 at this point in the process isreferred to as the collected bone marrow composition.

[0066] At this point in the method, or earlier, it may be desirable totest or sample the composition in the first container 11. In thisembodiment of the invention, a sampling device (not illustrated) may beinserted through re-sealable sampling port 40 and into the compositioncontained therein. Once a sample is withdrawn from the container,sampling port 40 preferably closes or re-seals.

[0067] The collected bone marrow may then be processed by opening orestablishing fluid communication between the first container 11 and thesecond container 14. By creating a pressure differential between firstcontainer 11 and second container 14, biological fluid will flow fromthe first container to the second container. In accordance with someembodiments of the invention, the bone marrow will be filtered asdesired if a filter assembly is interposed in the fluid flow pathbetween the first container 11 and the second container 14.

[0068] The invention also involves a method for harvesting, collecting,and processing bone marrow comprising harvesting bone marrow in acontainer, collecting bone marrow in a collection container; and passingthe bone marrow through a filter assembly and into a second container.

[0069] In general, donated bone marrow is processed as soon aspracticable in order to more effectively reduce or eliminatecontaminating factors, including but not limited to leucocytes andmicroaggregates.

[0070] Each of the components of the assembly will now be described inmore detail below.

[0071] The containers which are used in the biological fluid processingassembly may be constructed of any material compatible with a biologicalfluid, such as bone marrow or a bone marrow component, and is capable ofwithstanding a sterilization environment. A wide variety of thesecontainers are already known in the art. For example, bone marrowcollection and satellite bags are typically made from plasticizedpolyvinyl chloride, e.g. PVC plasticized with dioctylphthalate,diethylhexylphthalate, or trioctyltrimellitate. The bags may also beformed from polyolefin, polyurethane, polyester, and polycarbonate.

[0072] As used herein, the tubing may be any conduit or means whichprovides fluid communication between the containers, and is typicallymade from the same flexible material as is used for the containers,preferably plasticized PVC. The tubing may extend into the interior ofthe container, and may be used as a siphon, for example. There may be anumber of tubes providing fluid communication to any individualcontainer, and the tubes may be oriented in a number of ways. Forexample, there may be at least two tubes oriented at the top of thecollection bag, or at the bottom of the bag, or a tube at each end ofthe bag.

[0073] Additionally, the tubes, assemblies, porous media, andcontainers, may be oriented to define different flow paths. For example,when bone marrow is processed, a first portion may flow along a firstflow path, e.g., through a 850 micron filter and into a satellite bag(e.g., a second container). Similarly, a second portion may flow along asecond flow path, e.g., through a 200 micron filter assembly, and into asatellite bag (e.g., a third container). Since independent flow pathsmay be present, biological fluids may flow concurrently, orsequentially.

[0074] A seal, valve, clamp, transfer leg closure, or the like istypically located in or on the tubing. It is intended that the presentinvention is not limited by the type of material used to construct thecontainers or the conduit which connects the containers.

[0075] As noted above, the bone marrow is passed through one or moreporous media adapted to remove one or more predetermined constituents inthe bone marrow. As used herein, adapted to remove refers to a physical,biological, chemical, or other arrangement or surface enhancementintended to effect the removal of the component. For example, it may bedesirable to remove all components in the bone marrow having a sizegreater than about 850 microns. In this embodiment, the porous mediawould be adapted to remove particles greater than about 850 microns byhaving a pore rating of about 850 microns. In a second example, it maybe desirable to remove leukocytes from the bone marrow. In thisembodiment, the porous media might be adapted to remove leukocytes bycoating the surface of a fibrous porous media with one or more elementsthat attract and/or bind leukocytes. In a third example, it may bedesirable to specifically remove stem cells from the bone marrow. Inthis embodiment, the porous media might be adapted to remove stem cellsby coating the surface of a fibrous porous medium with an antibody orthe like that specifically binds stem cells. It is intended that thepresent invention should be limited to a specific configuration of theporous medium.

[0076] The porous medium may be formed from any natural or syntheticfiber (or from other materials of similar surface area and pore size)compatible with blood. The porous medium may remain untreated.Preferably, the critical wetting surface tension (CWST) of the porousmedium is within a certain range as dictated by its intended use and/orby the type of biological fluid passing through the porous medium. Thepore surfaces of the medium may be modified or treated in order toachieve a desired property. Although the filter medium may remainuntreated, the fibers or membrane are preferably treated to make themeven more effective for separating one component from other componentsof bone marrow. The porous medium is preferably treated in order toreduce or eliminate platelet adherence to the medium. Any treatmentwhich reduces or eliminates platelet adhesion is included within thescope of the present invention. Furthermore, the medium may be surfacemodified as disclosed in U.S. Pat. No. 4,880,548, incorporated herein byreference, in order to increase the critical wetting surface tension(CWST) of the medium and to be less adherent of platelets.

[0077] In the embodiments of the invention in which the porous mediumincludes fibers, the surface characteristics of the fiber may remainunmodified, or can be modified by a number of methods, for example, bychemical reaction including wet or dry oxidation; by coating the surfaceby depositing a polymer thereon; or by grafting reactions wherein thesubstrate or fiber surface is activated prior to or during wetting ofthe fiber surface by a monomer solution by exposure to an energy sourcesuch as heat, a Van der Graff generator, ultraviolet light, or tovarious other forms of radiation; or by subjecting the fibers to gasplasma treatment. A preferred method is a grafting reaction usinggammaradiation, for example, from a cobalt source.

[0078] An exemplary radiation grafting technique employs at least one ofa variety of monomers each comprising an ethylene or acrylic moiety anda second group, which is preferred to be a hydrophilic group (e.g.,—COOH, or —OH). Grafting of the fibrous medium may also be accomplishedby compounds containing an ethylenically unsaturated group, such as anacrylic moiety, combined with a hydroxyl group, preferably monomers suchas hydroxyethyl methacrylate (HEMA), or acrylic acid. The compoundscontaining an ethylenically unsaturated group may be combined with asecond monomer such as methyl acrylate (MA), methyl methacrylate (MMA),or methacrylic acid (MAA). Analogues with similar functionalcharacteristics may also be used to modify the surface characteristicsof fibers.

[0079] If desired, the flow rate of biological fluid through the filtercan be regulated to obtain a total flow period of about 10 to 40 minutesby selecting the appropriate element diameter, element thickness, fiberdiameter, and density, and/or by varying the diameter of the tube eitherupstream or downstream of the filter, or both up and downstream.

[0080] All of these parameters can be varied; for example, the diameterof the porous medium could be reduced and the thickness increased whileretaining the same total quantity of fiber, or the fibers could belarger in diameter while increasing the total quantity of fiber, or thefibers could be packed as opposed to preformed into a cylindrical disc.Such variations fall within the purview of this invention.

[0081] As noted above, as the bone marrow is expressed from thecollection bag, it may be processed through a device having a leucocytedepletion element in order to reduce the leucocyte content of the bonemarrow. In accordance with the invention, the porous medium for removingleucocytes from the bone marrow may comprise a leucocyte removal elementor porous medium having an average diameter of from about 1 to about 4μm, preferably from about 2 to about 3 μm. Polybutylene terephthalate(PBT) web, which is a preferred material, may be hot compressed to avoids volume of about 65% to about 90% and preferably to about 73% toabout 88.5%.

[0082] The biological fluid may be supplied in any suitable quantityconsistent with the capacity of the overall device and by any suitablemeans, e.g., in a batch operation by, for example, a collection bagconnected to an expresser or a syringe, or in a continuous operation aspart of, for example, an apheresis system. A source of biological fluidmay also include an apheresis system, and/or may include a system inwhich biological fluid is re-circulated through the system.

[0083] Further, a bone marrow system according to the invention mayinclude an access port 12 configured as or in fluid communication with amanifold valve, said manifold valve having one or more ports adapted forcommunicating with a syringe. In this embodiment of the invention, bonemarrow may be harvested by multiple technicians using multiple syringes,and the bone marrow in multiple syringes may be transferred to thecollection bag simultaneously or consecutively.

[0084] The present inventive device may similarly be part of anapheresis system. The biological fluid to be processed may be handled ineither a batch or continuous manner. The sizes, nature, andconfiguration of the present inventive device can be adjusted to varythe capacity of the device to suit its intended environment.

[0085] Under certain circumstances, it may be desirable to maximize therecovery of a biological fluid retained or entrapped in various elementsof the biological fluid processing system. For example, under typicalconditions, using a typical device, the biological fluid will drainthrough the system until the flow is stopped, leaving some of the fluidin the system. In one embodiment of the invention, the retained fluidmay be recovered by using at least one gas inlet and/or at least one gasoutlet.

[0086] The invention may also comprise at least one gas inlet, and/or atleast one gas outlet. For example, first container 11 may also be influid communication with a gas inlet, a microaggregate filter assembly15, and a gas outlet. The assembly may also include additionalcontainers, flow paths, and porous media. The gas outlet is a porousmedium which allows gas that may be present in a biological fluidprocessing system when the biological fluid is processed in the system,out of the system. The gas inlet is a porous medium which allows gasinto a biological fluid processing system.

[0087] As used herein, gas refers to any gaseous fluid, such as air,sterilized air, oxygen, carbon dioxide, and the like; it is intendedthat the invention is not to be limited to the type of gas used.

[0088] The gas inlet and gas outlet are chosen so that the sterility ofthe system is not compromised. The gas inlet and the gas outlet areparticularly suited for use in closed systems, or may be used later, forexample, within about 24 hours of a system being opened.

[0089] The gas inlet and the gas outlet each comprise at least oneporous medium designed to allow gas to pass therethrough. A variety ofmaterials may be used, provided the requisite properties of theparticular porous medium are achieved. These include the necessarystrength to handle the differential pressures encountered in use and theability to provide the desired filtration capability while providing thedesired permeability without the application of excessive pressure. In asterile system, the porous medium should also preferably have a porerating of about 0.2 micrometer or less to preclude bacteria passage.

[0090] The gas inlet and gas outlet may comprise a porous medium, forexample, a porous fibrous medium, such as a depth filter, or a porousmembrane or sheet. Multilayered porous media may be used, for example, amultilayered microporous membrane with one layer being liquophobic andthe other liquophilic. Particularly preferred are skinless,substantially alcohol-insoluble, hydrophilic polyamide membranes, suchas those described in U.S. Pat. No. 4,340,479.

[0091] The rate of air flow through the gas outlet or the gas inlet canbe tailored to the specific biological fluid or fluids of interest. Therate of air flow varies directly with the area of the porous medium andthe applied pressure. Generally, the area of the porous medium isdesigned to enable the biological fluid processing system to be primedin a required time under the conditions of use. For example, in medicalapplications it is desirable to be able to prime an intravenous set infrom about 30 to about 60 seconds. In such applications as well as inother medical applications, the typical porous medium is a membrane,which may be in the form of a disc which has a diameter from about 1 mmto about 100 mm, preferably from about 2 mm to about 80 mm, and morepreferably from about 3 mm to about 25 mm.

[0092] In accordance with the invention, the processing system may beprovided with a gas inlet to permit the introduction of gas into thesystem, and/or with a gas outlet to permit gases in the various elementsof the system to be separated from the biological fluid to be processed.The gas inlet and the gas outlet may be used together in connection withat least one assembly, porous medium, or container in the system, orthey may be used separately.

[0093] To that end, a gas inlet or gas outlet may be included in any ofthe various elements of the biological fluid processing system. By wayof illustration, a gas inlet or gas outlet may be included in at leastone of the conduits which connect the different containers, in a wall ofthe containers that receive the processed biological fluid, or in a porton or in one of those containers. The gas inlet or gas outlet may alsobe included on or in a combination of the elements mentioned above.Also, an assembly or porous medium may include one or more gas inlet orgas outlet as described above. Generally, however, it is preferred toinclude a gas inlet or gas outlet in the conduits which connect thecontainers or in the functional medical device. Included within thescope of the invention is the use of more than one gas inlet or gasoutlet in any conduit, receiving container, assembly, or porous medium.

[0094] It will be apparent to one skilled in the art that the placementof a gas inlet or a gas outlet may be optimized to achieve a desiredresult. For example, it may be desirable to locate the gas inletupstream of a porous medium and in or as close to the first container asis practical in order to maximize the recovery of biological fluid.Also, it may be desirable to locate the gas outlet downstream of theporous medium and as close to the receiving container as is possible inorder to maximize the volume of gas that is removed from the system.

[0095] Such placement of the gas inlet or gas outlet is particularlydesirable where there is only one gas inlet or gas outlet in the system.

[0096] In accordance with the invention, recovery from the variouselements of the biological fluid processing system may be maximized. Forexample, bone marrow is subjected to a processing step, resulting infiltered bone marrow. Blood product that has become entrapped in theseelements during processing may be recovered either by passing purge gasthrough the conduits and porous media, or by creating at least a partialvacuum in the system to draw out the retained blood product and topermit it to drain into the appropriate receiving container or assembly.

[0097] The purge gas may be from any of a number of sources. Forexample, the biological fluid processing system may be provided with astorage container for the storage of the purge gas, the purge gas may bethe gas that was removed from the system during the processing function,or the purge gas may be injected aseptically into the system from anoutside source (e.g., through a syringe). For example, it may bedesirable to use sterile purge gas that has been sterilized in aseparate container apart from the biological fluid processing system.

[0098] In accordance with the invention, a clamp, closure, or the likemay be positioned on or in any or all of the conduits in order tofacilitate a desired function, i.e., establishing a desired flow pathfor biological fluid or gas.

[0099] The gases separated by the gas outlet may be vented from thesystem, or they may be collected in a gas container (not shown) andreturned to the system as a purge gas to facilitate the recovery ofbiological fluid that becomes trapped in the various components of thesystem.

[0100] After the system is primed and the gas outlet is inactivated, theclamp adjacent to the containers or assembly is opened to allow thecontainers to fill with processed biological fluid. This continues untilthe collection bag 11 collapses. In order to recover the very valuablebiological fluid retained in the system, ambient air or a sterile gasmay enter the system through gas inlet. If gas inlet is a manual inletmeans, a closure is opened or a clamp released; if the gas inlet isautomatic, the pressure differential between the gas inlet and thecontainers will cause the air or gas to flow through the conduits,through the porous media, and towards the respective containers. In theprocess, retained biological fluid that is trapped in those elementsduring processing are recovered from those components and collected inthe containers. It should be noted that the purge air or gas ispreferably separated from the biological fluid at gas outlet so thatlittle, if any, purge gas will be received by the containers. This maybe accomplished by clamping the conduit downstream of the gas outlet. Inanother embodiment of the invention, the purge air or gas may beseparated from the system through a gas outlet located in the bagitself.

[0101] A system according to the present invention may be used inconjunction with other assemblies or porous media, including filtrationand/or separation devices, e.g., a device for removing leucocytes from aplatelet-containing solution or concentrate.

[0102] A number of additional containers may be in communication withthe biological fluid processing system, and can be utilized to definedifferent flow paths. For example, an additional satellite bagcontaining physiological solution may be placed in communication withthe biological fluid processing system upstream of the filter assembly,and the solution may be passed through the filter assembly so that thebiological fluid that was held up in the assembly can be collected.

[0103] It will be appreciated that when the biological fluid from thecollection bag 11 is expressed towards the containers, some of thebiological fluid may be trapped in the conduits and/or the porousmediums. For example, 8 cc to 35 cc is typically retained in the system;but as little as 2 cc to as much as 150 cc or more may be retained insome types of systems.

[0104] In an embodiment of the invention (not shown), air or gas may bestored in at least one gas container; upon opening of valve or clampmeans in the conduits, gas can be fed through them to purge the conduitsand assemblies, thereby facilitating the recovery of biological fluidthat may have been trapped during processing.

[0105] Preferably, the purge air or gas is fed to the conduits at apoint as close as is reasonably possible to container 11 to maximize thevolume of biological fluid recovered. The air or gas container ispreferably flexible so that the gas therein may be fed to the systemmerely by simple compression. The biological fluid containers and theair or gas containers may be composed of the same material.

[0106] In an embodiment of the invention (not shown), priming fluid maybe stored in at least one container; upon opening of valve or clampmeans in the conduits, priming fluid can be fed through them to primeand/or wet the conduits and assemblies, thereby facilitating thefunction and operation of the various elements in the system. Primingfluid includes but is not limited to a solution or compositioncontaining bone marrow, an anti-coagulant solution, or any solutioncompatible with bone marrow and effective as a wetting or primingsolution.

[0107] It will be apparent to one skilled in the art that the placementand connection of a source of priming fluid to the system may beoptimized to achieve a desired result.

[0108] Priming, as used herein, refers to wetting or priming the innersurfaces of a device or assembly prior to its actual use allowing aseparate assembly to be injected into the system. A valve or clamp isopened to allow fluid to flow through the assembly; then, with thepassage of fluid through the assembly, gas downstream of the fluid isexpelled through the gas outlet until fluid reaches a branching element,at which point the clamp is closed. With the clamp in a closed position,the connector downstream of the gas outlet may be opened or readied foruse without fluid in the assembly dripping through the connector.

[0109] In accordance with the invention, the biological fluid collectionand processing assembly should be able to withstand rigoroussterilization, typically consisting of radiation sterilization (at about2.5 megarads), and/or autoclaving (at about 110° C. to about 120° C. forabout 15 to 60 minutes).

[0110] There may be a clamp or the like between the collection bag 11and the flexible tubing or within the tubing, to prevent the flow of thebone marrow from entering the wrong conduit.

[0111] Movement of the biological fluid through the system is effectedby maintaining a pressure differential between the source of thebiological fluid and the destination of the biological fluid (e.g., acontainer such as a satellite bag or a needle on the end of a conduit).The system of the invention is suitable for use with conventionaldevices for establishing the pressure differential, e.g., an expresser.Exemplary means of establishing this pressure differential may be bygravity head, applying pressure to the collection bag (e.g., by hand orwith a pressure cuff), or by placing the other container (e.g.,satellite bag) in a chamber (e.g., a vacuum chamber) which establishes apressure differential between the collection bag and the othercontainer. Also included within the scope of the invention may beexpressors which generate substantially equal pressure over the entirecollection bag.

[0112] While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth in the Examples. Itshould be understood that these specific embodiments are not intended tolimit the invention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

What is claimed is:
 1. A bone marrow collection and processing systemcomprising: a first container, said first container having at least oneaccess port and at least one discharge port; a second containercommunicating with the first container; at least one porous mediuminterposed between the first container and the second containercomprising at least one size depletion medium.
 2. The bone marrowcollection and processing system of claim 1 wherein the first containeris sealable.
 3. The bone marrow collection and processing system ofclaim 1 wherein the porous medium is a microaggregate filter.
 4. Thebone marrow collection and processing system of claim 3 wherein themicroaggregate filter has a pre-determined pore rating.
 5. The bonemarrow collection and processing system of claim 1 further comprising athird container in fluid communication with the first container.
 6. Thebone marrow collection and processing system of claim 5 furthercomprising a manifold interposed in the fluid flow path between thefirst container and the third container.
 7. The bone marrow collectionand processing system of claim 6 further comprising a manifold having aport adapted for engagement with a bone marrow harvesting container. 8.A bone marrow collection and processing system comprising a first sealedcontainer, said first container having at least one access port and atleast one discharge port; and at least one second sealed container influid communication with the first container.
 9. A method for collectingand processing bone marrow comprising: harvesting bone marrow from adonor; collecting the bone marrow in a sealed collection container;passing a portion of the bone marrow through a first porous medium, thefirst porous medium comprising a size gradient filter; and collectingthe filtered bone marrow in a second container.
 10. A method forcollecting and processing bone marrow comprising transferring harvestedbone marrow into a sealed first container; processing the harvested bonemarrow; and passing the processed bone marrow into a second container.11. The method of claim 10 wherein processing the harvested bone marrowincludes filtering the bone marrow.
 12. The method of claim 10 whereintransferring harvested bone marrow into a sealed first containerincludes passing the harvested bone marrow through at least onemanifold.
 13. The method of claim 12 further comprising passing asolution housed in a third container through the manifold.
 14. Themethod of claim 13 further comprising passing the solution through themanifold into harvesting container in fluid communication with themanifold, and then returning the solution to the third container. 15.The method of claim 14 further comprising passing the solution in thethird container into the first container.
 16. The method of claim 10further comprising priming the first container and the second container.17. The method of claim 11 further comprising priming the firstcontainer and the second container.
 18. The method of claim 12 furthercomprising priming the manifold.